JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 JPET FastThis article Forward. has not beenPublished copyedited on and January formatted. The8, 2004final version as DOI:10.1124/jpet.103.059394 may differ from this version.

JPET #59394

Salvinorin A, an Active Component of the Hallucinogenic Sage Salvia divinorum, is a Highly Efficacious Kappa Opioid Receptor Agonist: Structural and Functional

Considerations1

Downloaded from

Charles Chavkin, Sumit Sud, Wenzhen Jin, Jeremy Stewart, Jordan K. Zjawiony, Daniel

J. Siebert, Beth Ann Toth, Sandra J. Hufeisen and Bryan L. Roth jpet.aspetjournals.org

Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195 (CC, SS, WJ); Salvia divinorum Research and Education Center, P.O. Box 661552. Los Angeles, CA 90066 USA (DS); Department of Pharmacognosy, at ASPET Journals on September 27, 2021 University of Mississippi, University, MS 38677-1848 (JS, JKZ); NIMH Psychoactive Drug Screening Program and Department of Biochemistry, Case Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106 (BAT, SJH, BLR)

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version.

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Running title: Salvinorin A activates κ- Opioid Receptors

Address correspondence to:

Bryan Roth MD, PhD Department of Biochemistry; RM RT500-9 Case Western Reserve University Medical School 2109 Adelbert Road Cleveland, OH 44106 216-368-2730 (Office); 216-368-3419 (Fax) [email protected] (email) Downloaded from Text pages: 13

Tables: 2

Figures: 5 jpet.aspetjournals.org

References: 24

at ASPET Journals on September 27, 2021 Abstract: 240 words

Introduction: 246 words

Discussion: 788 words

Abbreviations:

Salvinorin A (Sal A), dynorphin A (Dyn A), norbinaltorphimine (nBNI);

U50,488, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate; U69,593, (+)-(5α,7α,8β)-N-Methyl-N-[7-

(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide.

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ABSTRACT

The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high affinity and selective κ-opioid receptor agonist (Roth et al, 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human κ-opioid receptors expressed in HEK-293 cells.

Thus, salvinorin A, salvinorinyl-2-propionate and salvinorinyl-2-heptanoate were found Downloaded from to be either full (salvinorin A) or partial (2-propionate; 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist jpet.aspetjournals.org potency and efficacy of salvinorin A, performed by co-transfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked

intracellular calcium mobilization, affirmed that Salvinorin A was a potent and effective at ASPET Journals on September 27, 2021

κ-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2 position of salvinorin A was critical for κ-opioid receptor binding and activation. Since issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein gated K+ channels co-expressed in Xenopus oocytes—a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than U50488 or U69593 (two standard κ-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for κ-opioid receptors). Salvinorin A thus represents the first known naturally-occurring non-nitrogenous full agonist at κ-opioid receptors.

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Salvia divinorum, a member of the Lamiaceae family, has been used by the

Mazatec Indians of northeastern Oaxaca, Mexico primarily for its psychoactive effects

(Wasson, 1962; Wasson, 1963) for many hundreds of years (see Valdes et.al., 1983;

Sheffler and Roth, 2003 for reviews). The active ingredient of Salvia divinorum is salvinorin A, a non-nitrogenous neoclerodane diterpene which represents the most potent

naturally occurring hallucinogen known (Valdes et al., 1984) (Siebert, 1994). Salvinorin Downloaded from

A induces an intense, short-lived hallucinogenic experience qualitatively distinct from that induced by the classical hallucinogens lysergic acid diethylamide (LSD), psilocybin jpet.aspetjournals.org and mescaline (Siebert, 1994). Both Salvia divinorum and salvinorin A have been used recreationally for their hallucinogenic properties (Giroud et al., 2000). Intriguingly, an anecdotal case report has suggested that Salvia divinorum may have antidepressant at ASPET Journals on September 27, 2021 properties as well (Hanes, 2001).

Quite recently we discovered that salvinorin A has high affinity and selectivity for the cloned κ- opioid receptor (KOR) and suggested, based on limited functional studies, that salvinorin A was a ΚΟR agonist (Roth et al., 2002). We now present a detailed report on the agonist properties of salvinorin A and selected derivatives. We discovered that salvinorin A is an extrodinarily efficacious and potent κ-opioid agonist. We also found, based on structure-function studies, that the nature of the substituent on the 2- position of salvinorin has profound affects functional activity. Taken together, these results support the hypothesis that the unique effects of salvinorin A on human perception are due to selective activation of KOR.

4 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version.

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Materials and Methods

Materials

U50488, U69593, dynorphin A, norbinaltorphimine (nBNI) were obtained from Sigma Chemical Co. (St Louis, MO). [3H]-bremazocine was from New England Nuclear (Boston, MA).

Complementary DNA Clones and cRNA Synthesis for oocyte studies

The rat KOR was obtained from Dr. David Grandy (GenBank accession number Downloaded from

D16829). The human KOR cDNA was obtained from the Guthrie Rsearch Foundation

(GenBank accession number NM000912) and subcloned into the eukaryotic expression jpet.aspetjournals.org vector pIRESNEO (Invitrogen Inc); cDNAs for KIR3.1 (accession number U01071) and

KIR3.2 (accession number U11859) were obtained from Drs. Cesar Lebarca and Henry

Lester, respectively. The chimeric G protein Gq-i5 was obtained from Bruce Conklin at ASPET Journals on September 27, 2021

(University of California, San Francisco), while Gα16 was obtained from the Guthrie

Research Foundation; both constructs were verified by automated dsDNA sequencing

(Cleveland Genomics, Inc) prior to use. Plasmid templates for all constructs were linearized prior to cRNA synthesis, and the mMESSAGE MACHINE kit (Ambion Inc.) was used to generate capped cRNA.

Cell lines and maintenance

A stable line expressing the human KOR (hKOR-293) was obtained by transfecting an hKOR expression vector (hKOR-pIRESNEO) into HEK-293 cells

(maintained and transfected as previously detailed (Roth et al., 2002)) and selecting in

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600 µg/ml G418. Surviving clones were expanded and characterized with one (hKOR-

293) which expressed high levels of hKOR (ca 1 pmol/mg) used for further studies.

Oocyte maintenance and injection

Healthy stage V and VI oocytes were harvested from mature anesthetized

Xenopus laevis (Nasco, Ft Atkinson, WI, USA) and defolliculated enzymatically as

described previously (Snutch, 1988). The oocytes were maintained at 18˚C in standard Downloaded from oocyte buffer, ND96 (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM

HEPES, pH 7.5), supplemented with 2.5 mM sodium pyruvate and 50 µg/ml gentamicin jpet.aspetjournals.org (Sigma Chemical, St Louis, MO, USA). One day after harvest, cRNAs were injected (50 nl/oocyte) with a Drummond microinjector. Each oocyte was injected with 0.5 ng of KOR cRNA and 0.1 ng of KIR3.1 and KIR3.2 cRNA. Recordings were made at least 48 h after at ASPET Journals on September 27, 2021 injection.

Electrophysiological studies

An Axon Geneclamp 500 amplifier was used for standard two-electrode voltage- clamp experiments. The FETCHEX program (Axon Instruments, Foster City, CA) and recorded data traces were used for data acquisition and analysis. Oocytes were then removed from incubation medium, placed in the recording chamber containing ND96 medium, and clamped at –80mV. Recordings were made in hK buffer (72.5 mM NaCl,

24 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5). To facilitate the

+ recording of inward K currents through the KIR3 channels, the normal oocyte saline buffer was modified to increase the KCl concentration to 24 mM K+. Microelectrodes were filled with 3 M KCl and had resistances of 0.4-2.0 MΩ.

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Radioligand binding and functional studies

Radioligand binding studies were performed as previously described (Roth et al.,

2002) with the exception that 150 mM NaCl was added to the standard binding buffer to mimic physiological sodium concentrations. In brief, membranes (10-50 ug) were incubated together with 3H-bremazocine in a final volume of 0.5 ml with a buffer of the following composition: 50 mM Tris-HCl, 150 mM NaCl, pH=7.40 along with test agents

for 90 min at room temperature. Incubations were terminated by rapid filtration and Downloaded from collection on GF/C glass fiber filters and washing with ice-cold binding buffer. Dried filters were put into sample vials, scintillation fluid added and dpm measured by liquid jpet.aspetjournals.org scintillation spectroscopy. Measurements of the ability of KOR agonists to inhibit forksolin-stimulated adenylate cyclase activity were performed as detailed previously

(Roth et al., 2002). For studies involving measurements of intracellular calcium at ASPET Journals on September 27, 2021 mobilization, a Molecular Devices FLEXSTATION was used as recently detailed

(Rothman et al, JPET, in press). For these studies hKOR were co-transfected with the chimeric G protein Gaq-i5 (Conklin et al., 1993) or the ‘universal’ G protein Ga16

(Offermanns and Simon, 1995). Measurements of intracellular calcium mobilization and quantification of agonist efficacy and potency were performed as recently described

(Rothman et al, JPET, in press).

Data Analysis

EC50 values and curve fitting were determined using Nfit software (Island

Products, Galveston, TX) or GraphPad Prizm. Student's t test was used for comparison of independent means, with values reported as two-tailed p values.

7 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version.

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Chemistry.

Salvinorin A was isolated from dried leaves of Salvia divinorum by the method reported earlier (Valdes et al., 1984). Salvinorin A was hydrolyzed using potassium carbonate in methanol to yield salvinorin B. The reported esters were formed using salvinorin B, dimethylaminopyridine, and the corresponding acid chloride in methylene

chloride. Downloaded from

Salvinorin B was characterized by 1H-NMR, 13C-NMR, and high-resolution mass spectrometry (HRMS) and found to be authentic by comparison with literature values jpet.aspetjournals.org (Valdes et al., 1984). The reported esters were purified by HPLC and characterized by

HRMS. 1H and 13C NMR spectra were recorded on a Bruker AMX 500 MHz NMR spectrometer in CDCl3. The HRMS were measured using a Bioapex FT mass at ASPET Journals on September 27, 2021 spectrometer with electrospray ionization. HPLC was conducted on a Waters Deltaprep

4000 system using a Waters Xterra RP18, 5 µm, 4.6 mm x 150 mm column, with mobile phase H2O/Acetonitrile (1:1). TLC analyses were carried out on pre-coated Si gel G254,

250 µm plates, with the developing system hexane/EtOAc (2:1) and visualized with vanillin/H2SO4 in EtOH.

Preparation of Esters. Salvinorin B (10mg, 26 nmol) and 4-dimethylaminopyridine

(catalytic amount) were dissolved in methylene chloride (3 ml). The corresponding acid chloride (130 nmol) was added, and the reaction stirred at room temperature overnight.

The mixture was quenched with methanol, loaded onto silica, and purified by vacuum

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JPET #59394 liquid chromatography using Si gel (230-400 mesh) with hexane/EtOAc (3:1) solvent system. Calculated molecular weights were obtained using ChemDraw software.

Yields and Masses of Salvinorinyl Esters Calculated Found(M + 23) for sodium

1) propionate 9.0 mg, 78.5% 446.1941 469.1917

2) heptanoate 10.5 mg, 81.6% 502.2567 525.2566

3) pivalate 11.1 mg, 91.4% 474.2254 497.2215 Downloaded from

4) p-bromobenzoate 12.4 mg, 84.4% 572.1046 595.1009

5) 2,2,2-trichloroethylcarbonate 11.5 mg, 79.4% 564.0721 587.0689 jpet.aspetjournals.org 6) ethylcarbonate 9.8 mg, 82.7% 462.1890 485.1833

7) piperonylate 1.6 mg, 11.6% 538.1839 561.1834

8) 1-naphthoate 2.1 mg, 15.1% 544.2097 567.2087 at ASPET Journals on September 27, 2021

9) cyclopropanecarboxylate 10.5 mg, 89.4% 458.1941 481.1952

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Results

In initial studies, we examined the abilities of salvinorin A and selected derivatives (see Fig 1 for structures) for their ability to bind to hKORs. As can be seen, the synthetic derivatives of salvinorin A differ solely in the nature of the substituent in the 2-position. As is shown in Table I, salvinorinyl-2-propionate was the only derivative with sub-micromolar affinity for hKOR’s; also of note is that salvinorin B was

inactive at hKORs. A screen of a number of other receptor subtypes showed that the Downloaded from salvinorin A derivatives tested had no significant activity at other receptors including various serotonergic, dopaminergic, muscarinic, adrenergic, cannabinoid, and sigma jpet.aspetjournals.org receptors (Table I for details)

We next evaluated the ability of salvinorin A and the propionate and heptanoate derivatives to activate hKOR’s by measuring the ability to inhibit forskolin-stimulated at ASPET Journals on September 27, 2021 cAMP production using U69593 as the comparator. As shown in Table I, salvinorin A and salvinorinyl-2-propionate were potent and full agonists when compared with U69593 while salvinorinyl-2-heptanoate was a partial agonist.

We also evaluated the ability of U69593, dynorphin A, salvinorin A and the propionate derivative of salvinorin A to activate hKOR’s using a fluorescent-microplate- reader (FLEXSTATION) wherein hKOR’s were co-transfected with either the chimeric

G-protein Gqi5 or the universal G protein Ga16 as previously detailed (Rothman et al, in press). Fig 2 shows representative results for U69593 and salvinorin A using either

Gα16 (2A,B) or Gq-i5 (2C,D). No responses were seen in untransfected cells or in cells transfected with hKOR alone (not shown). Fig 2 also shows a representative dose- response study using Gq-i5 as the chimeric G protein. Since both methods appeared to

10 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #59394 yield equivalent results, further studies were performed with Gq-i5. Table II shows representative EC50 and Emax values for a variety of KOR agonists using Gq-i5. In these studies, salvinorin A was more potent than any other of the tested KOR agonists (Table

II). In terms of maximal response, all of the active compounds gave similar responses .

It is well known that over-expression systems tend to provide inaccurate estimates of agonist potencies and efficacies because of issues of receptor reserve (Kenakin, 2002).

As well, it has been well-described that unnatural expression systems wherein chimeric Downloaded from or ‘universal’ G proteins are used also lead to misleading estimates of agonist potencies and maximal responses (Woolf et al., 2001) (Kenakin, 2002). Accordingly, we next jpet.aspetjournals.org determined the maximal agonist responses (Emax ) and potencies (EC50s) of selected compounds using a system without receptor reserve.

at ASPET Journals on September 27, 2021

Salvinorin A is a full agonist. For these studies, Xenopus oocytes were co-injected with inwardly rectifying K+-channels and KORs. In the experiment shown, a representative oocyte voltage clamped at –80 mV was first perfused with hK buffer (containing 24 mM

KCl) to shift the reversal potential of potassium and facilitate K+ current through Kir3

(Fig 3). Perfusion with 1 µM salvinorin A significantly increased the inward current, and the activation was reversed by 100 nM norBNI. Similarly, 1 µM U69593 increased the inward current in a different oocyte, and the effect was also blocked by 100 nM norBNI

(Fig 1B). Neither 10 µM salvinorin A nor U69593 increased the membrane conductance of oocytes expressing Kir3 without KOR (data not shown).

Concentration-response curves of salvinorin-A, and kappa agonists U69593 and

U50488 were compared (Fig 4). Each point represents the mean response measured in 4-

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7 different oocytes. Data were collected from multiple batches of oocytes and merged by normalizing the responses to the average maximal response produced by salvinorin A on that recording day. Based on these results, salvinorin A was not significantly more potent

(EC50 = 69 nM; Confidence intervals: 50-94 nM) than U69593 (EC50 = 224 nM;

Confidence intervals: 51-157 nM) or U50488 (EC50 150 nM; Confidence intervals: 50-

194nM).

Under these expression conditions, there was an apparent lack of spare kappa Downloaded from receptors. Increasing the kappa receptor cRNA from 0.5 ng/oocte to 1.0 ng increased the average U69593 response from 1.63 ± 0.57 µA to 2.76 ± 1.04 µA (n=7,8). Based on the jpet.aspetjournals.org lack of spare receptors, we directly compared the maximal responses evoked by 10 µM each of the kappa agonists (Fig 5) with that of Dynorphin A. In this assay Propionyl-

Salvinorin also acted as a partial agonist whose maximal activity was less than Salvinorin at ASPET Journals on September 27, 2021

A. The response to salvinorin A was significantly greater than that to U69593 and

U50488 (p<0.05), but not significantly greater to that of dynorphin A.

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Discussion

The principal finding of this study is that salvinorin A is an extraordinarily potent full agonist at hKORs. Additionally, we report that salvinorinyl-2-propionate is a potent partial agonist at KOR’s and also demonstrate that the nature of the 2-substituent of the salvinorin scaffold is critically important for agonist efficacy and potency. We also have obtained data with KOR-knock-out and wild-type mice that the actions of salvinorin A

are mediated by KOR in vivo (Pintar et al, manuscript in preparation). Taken together, Downloaded from these results imply that the profound effects of salvinorin A on human consciousness are mediated by potent and highly efficacious activation of KORs. jpet.aspetjournals.org In prior reports we have suggested that because salvinorin A is a potent hallucinogen which is apparently selective for KORs, that targeting KORs might lead to novel medications for the treatment of diseases manifested by hallucinatory experiences at ASPET Journals on September 27, 2021

(e.g. schizophrenia, affective disorders, dementia) (Roth et al., 2002) (Sheffler and Roth,

2003). In this regard, studies with non-selective opioid antagonists which possess KOR actions in schizophrenia have been mixed (Rapaport et al., 1993) (Sernyak et al., 1998), although there are no studies in which selective KOR antagonists have been tested.

Because of anecdotal reports that extracts of Salvia divinorum may possess antidepressant actions (Hanes, 2001) and published studies in rodents that KOR antagonists block stress-induced responses (McLaughlin et al., 2003), KOR antagonists could possess anti-anxiety/anti-depressant actions as well. Indeed, a recent study (Mague et al., 2003) suggested that kappa-selective antagonists might have intrinsic antidepressant actions. Our current studies suggest that novel KOR-selective agents

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JPET #59394 might be obtained by selective modification of the salvinorin scaffold. Whether such agents might possess antidepressant or antipsychotic activity is unknown.

As shown in these studies, salvinorin A and salvinorinyl-2-propionate are potent agonists at KORs with salvinorin A being a full agonist in most assay systems while salvinorinyl-2-propionate is likely a partial agonist. Salvinorin B and all other tested salvinorin derivatives were devoid of significant activity. One potential complication of

the studies performed on recombinant, over-expressed receptors relates to the issue of Downloaded from receptor reserve. Thus, it is widely appreciated that over-expressing G proteins and/or receptors in heterologous expression systems leads to inaccurate estimates of agonist jpet.aspetjournals.org potencies and maximal responses (for review see Kenakin, 1997). Accordingly, we also evaluated the agonist actions of salvinorin A and other compounds at KOR’s expressed in

Xenopus oocytes. at ASPET Journals on September 27, 2021

KOR expressed in Xenopus oocytes activate intrinsic G proteins that then increase the conductance of co-expressed G Protein Coupled Inwardly Rectifying potassium channels (GIRK, Kir3) (Henry et al, 1995). Injection of cRNAs coding for the mammalian receptor and channel has been demonstrated to faithfully reconstitute opioid signaling in oocytes equivalent to that observed in guinea pig substantia gelatinosa neurons (Grudt and Williams 1993). In addition, by controlling the levels of receptor and channel expression, spare receptors can be avoided and the peak responses produced by different drugs can be a direct measure of agonist efficacy. The in vitro bioassay also eliminates pharmacokinetic barriers, and the electrophysiological recording of channel activation provides a rapid measure of receptor activation. In this study, we compared the relative activity of salvinorin A with three compounds having established kappa opioid

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JPET #59394 receptor agonist activity. Salvinorin A was found to be more potent and have higher efficacy than either U50,4588 and U69593. The agonist efficacy of salvinorin A was not significantly different from dynorphin A(1-17), an endogenous neurotransmitter of the kappa opioid receptor (Chavkin et al., 1982).

Structure-activity relationship studies shows that KOR agonistic activity of salvinorin derivatives depend largely on the size and character of the substituent on the 2-

ester moiety. Generally, the studied derivatives have either lower affinity for KOR than Downloaded from salvinorin A or are completely devoid of activity. The two active derivatives, the propionate and the heptanoate, demonstrate that as the alkyl chain is lengthened, KOR jpet.aspetjournals.org affinity diminishes. Interestingly however, chain length must not be the only factor, because the short-chain ethylcarbonate derivative is absent of activity.

The current results support the conclusion that just as morphine is a natural plant at ASPET Journals on September 27, 2021 product able to activate the mu opioid receptor, salvinorin A is a natural plant product able to activate the KOR. The strongly psychotomimetic actions of salvinorin A suggest that the dynorphin/kappa opioid system may have a role in the regulation of cognition and perception and support earlier proposals that some forms of schizophrenic hallucinations may be caused by hyperactive endogenous opioid systems (Gunne et al.,

1977). Recent data implicating the KOR-dynorphinergic system in modulating stress and anxiety responses in rodents suggest that targeting KORs might also lead to novel anti- depressant and anxiolytic medications. Salvinorin A, by virtue of its potency, efficacy and selectivity as a KOR agonist will be an important tool for discovering the role that the KOR-dynorphinergic system has in health and disease.

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Figure legends

Figure 1. Structures of Salvinorin A, B, and 2-Salvinorinyl Esters. Shown are the structures of the compounds used in this study.

Figure 2. Salvinorin A mobilizes intracellular Ca++ when hKOR’s are co-transfected with the universal G protein G16 or the chimeric G protein. For these studies HEK-293 cells Downloaded from were transfected with hKOR and either Gqi5 or G16 and the mobilization of intracellular calcium quantified as previously described (Rothman et al, in press) using a 96-well FLEXSTATION. A and B show representative results with increasing doses of jpet.aspetjournals.org U69593 or salvinorin A (0, 10 and 100 nM) wherein hKOR’s were co-transfected with G16 while C and D show results obtained when hKOR’s were co-transfected with Gq-i5. E shows the average for N=3 separate experiments for dose-response studies to salvinorin A and U69593. at ASPET Journals on September 27, 2021

Figure 3. Salvinorin A is a highly efficacious kappa receptor agonist. Representative traces showing the change in current during a typical experiment. A large inward current was apparent as the K+ concentration was increased from 2 to 24 mM in normal oocyte saline buffer. Salvinorin A (1 µM) and U69593 (1 µM) in the buffer (24 mM K+) further increased Kir3 currents, and the response was reversed by nor-BNI (100 nM), a kappa antagonist.

Figure 4. Concentration-response curve of salvinorin A, and kappa agonists U69593 and U50488. Cumulatively higher concentrations of salvinorin A and the kappa agonists were applied to the bath. The agonist response at each concentration was normalized as a percentage of the maximal salvinorin A response. Each point represents the mean response measured in 4-7 different oocytes.

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Figure 5. Salvinorin A is more efficacious than U69593 and U50488 in kappa receptor- mediated activation of Kir3 currents. At saturating concentration, salvinorin A (10 µM) evoked a large Kir3 currents, which were significantly higher than the response evoked by U69593 (10 µM) or U50488 (10 µM). Data are mean ± SEM; **p < 0.05. Dynorphin A (10 µM) produced a response that was not significantly different from salvinorin A.

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jpet.aspetjournals.org at ASPET Journals on September 27, 2021

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1Footnote:

The work was supported by USPHS grant RO1 DA04123 from NIDA to CC and by the

NIMH Psychoactive Drug Screening Program and KO2MH01366 to BLR and by

RO1DA017204 to BLR.

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21 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on January 8, 2004 as DOI: 10.1124/jpet.103.059394 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021